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Hum. Reprod. Advance Access published online on September 20, 2009
Human Reproduction, doi:10.1093/humrep/dep334
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© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

In vitro post-meiotic germ cell development from human embryonic stem cells

B. Aflatoonian1,2,5, L. Ruban2,3, M. Jones1, R. Aflatoonian2,4, A. Fazeli2 and H.D. Moore1,2

1 Centre for Stem Cell Biology, Department of Biomedical Sciences, The University of Sheffield, Western Bank, Alfred Denny Building, Sheffield S10 2TN, UK 2 Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK

5 Correspondence address. E-mail: b.aflatoonian{at}shef.ac.uk

BACKGROUND: Investigating the mechanisms of human primordial germ cell (PGC) and gamete development are important for understanding the causes of infertility and effects of environmental chemicals on reproductive development. However, there are practical and ethical difficulties associated with obtaining human tissue in early development. The aim of this study was to investigate whether human embryonic stem cell-hESC-generated germ cells could provide an in vitro model of gamete development.

METHOD: Human ESCs were differentiated as embryoid bodies (EBs) in vitro. Gene and protein marker expression profiles of EBs in different periods of culture were analysed by quantitative polymerase chain reaction (Q-PCR) and immunolocalization to monitor germ cell development. Secretion of hormones involved in germ cell maturation was measured, to detect the existence of a germ cell niche within EBs.

RESULTS: Q-PCR revealed gene expression profiles consistent with PGC formation and germ cell development. A small population of post-meiotic spermatid cells were identified using sperm-specific antibodies (Protamine 1 and 1.97). Although gene expression profiles characteristic of oocyte development and follicle-like structures were detected, a committed oocyte with extra-cellular zona pellucida was not recognized with zona pellucida-specific monoclonal antibody.

CONCLUSIONS: hESCs can form PGCs and post-meiotic spermatids in vitro, however, there remains doubt about oocyte development. Levels of steroid hormones produced by EBs were significant when compared with known values for a similar quantity of human testis, suggesting that hESC may intrinsically create a favourable hormonal niche for spermatogenesis.

Key words: oocyte/germ cells/human embryonic stem cells/niche/sperm

3 Present address: Department of Biochemical Engineering, University College London, Roberts Building, Torrington Place, London WC1E 7JE, UK

4 Present address: Reproductive and Biomedicine Group, Medical school, Iran University of Medical Sciences, Tehran 14155-5983, Iran

Submitted on June 13, 2009; resubmitted on August 19, 2009; accepted on August 24, 2009.


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